PROJECT SUMMARY/ABSTRACT The iron overload disorder hereditary hemochromatosis is an endocrine liver disease that results from an inability to produce sufficient amounts of hepcidin, the iron-regulatory hormone produced by the liver. In hemochromatosis, increased absorption of dietary iron leads to the appearance of plasma non-transferrin-bound iron (NTBI), which is taken up by various tissues and cells leading to tissue iron overload and related pathology. Plasma NTBI is also commonly seen in the hematologic disease thalassemia major, an inherited blood disorder that requires regular blood transfusions, which over time result in iron overload. Although NTBI is the major contributor to tissue iron loading, our understanding of the molecular mechanisms that mediate NTBI uptake is incomplete. The primary long-term objective of this proposal is to define the proteins that transport iron into various tissues and cells, particularly those affected by iron-overload related pathology. Our central hypothesis is that the membrane transport proteins ZIP14 and ZIP8 participate in iron homeostasis and NTBI uptake. In the previous funding period we found that ZIP14 is the primary NTBI uptake mechanism in hepatocytes and pancreatic acinar cells, and that ZIP14 is required for iron loading of the liver and pancreas in mouse models of hemochromatosis and dietary iron overload. We also generated a variety of conditional Zip8 knockout mouse models to interrogate the roles of ZIP8 in iron metabolism and iron overload. In Aim 1 of the proposed research, we will continue to define the roles of ZIP14 in tissue iron loading by using ZIP14 knockout (Zip14-/-) mice intercrossed with hemojuvelin knockout (Hjv-/-) mice, a model of juvenile hemochromatosis. The current focus will be on endocrine organs including the anterior pituitary gland and adrenal gland. Using Hjv-/- mice, we will also assess the efficacy of reducing ZIP14 expression (pharmacologically or genetically) combined with iron chelation in mitigating tissue iron overload. In Aim 2, we will determine how ZIP14-mediated iron loading of pancreatic beta cells leads to beta cell dysfunction and diabetes. For this aim we generated a transgenic mouse model that overexpresses ZIP14 specifically in beta cells. When loaded with iron, the ZIP14 transgenic mice, similar to iron-loaded humans, accumulate iron in beta cells and develop diabetes. We will characterize the development of diabetes in these mice, focusing on changes that occur in beta cells. In Aim 3, we will continue to define the roles of ZIP8 in iron homeostasis, particularly its apparent role in stress erythropoiesis. To define the roles of ZIP8 in tissue iron loading, we will utilize inducible Zip8-/- mice as well as Zip8-/-;Zip14-/- double knockout mice, which will help to determine whether these two homologous proteins can compensate for each other.